RESUMO
Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3' ends of CRISPR-Cas guide RNAs1. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4 and PE5), edit types (substitutions, insertions and deletions), endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3' ends of RNA polymerase III transcripts2. We found that La functionally interacts with the 3' ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.
Assuntos
Edição de Genes , Proteínas de Ligação a RNA , Humanos , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Células K562 , Poli U/genética , Poli U/metabolismo , RNA Polimerase III/metabolismo , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
RNA binding proteins (RBPs) take part in all steps of the RNA life cycle and are often essential for cell viability. Most RBPs have a modular organization and comprise a set of canonical RNA binding domains. However, in recent years a number of high-throughput mRNA interactome studies on yeast, mammalian cell lines, and whole organisms have uncovered a multitude of novel mRNA interacting proteins that lack classical RNA binding domains. Whereas a few have been confirmed to be direct and functionally relevant RNA binders, biochemical and functional validation of RNA binding of most others is lacking. In this study, we used a combination of NMR spectroscopy and biochemical studies to test the RNA binding properties of six putative RBPs. Half of the analyzed proteins showed no interaction, whereas the other half displayed weak chemical shift perturbations upon titration with RNA. One of the candidates we found to interact weakly with RNA in vitro is Drosophila melanogaster end binding protein 1 (EB1), a master regulator of microtubule plus-end dynamics. Further analysis showed that EB1's RNA binding occurs on the same surface as that with which EB1 interacts with microtubules. RNA immunoprecipitation and colocalization experiments suggest that EB1 is a rather nonspecific, opportunistic RNA binder. Our data suggest that care should be taken when embarking on an RNA binding study involving these unconventional, novel RBPs, and we recommend initial and simple in vitro RNA binding experiments.
Assuntos
Proteínas de Drosophila/metabolismo , Proteínas Associadas à Distrofina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Tiorredoxinas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Sítios de Ligação , Clonagem Molecular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas Associadas à Distrofina/química , Proteínas Associadas à Distrofina/genética , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Modelos Moleculares , Ovário/citologia , Ovário/metabolismo , Poli U/química , Poli U/genética , Poli U/metabolismo , Ligação Proteica , RNA/química , RNA/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiorredoxinas/química , Tiorredoxinas/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Proteínas com Motivo Tripartido/química , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genéticaRESUMO
Cleavage and polyadenylation specificity factor 30 (CPSF30) is a zinc finger protein that regulates pre-mRNA processing. CPSF30 contains five CCCH domains and one CCHC domain and recognizes two conserved 3' pre-mRNA sequences: an AU hexamer and a U-rich motif. AU hexamer motifs are common in pre-mRNAs and are typically defined as AAUAAA. Variations within the AAUAAA hexamer occur in certain pre-mRNAs and can affect polyadenylation efficiency or be linked to diseases. The effects of disease-related variations on CPSF30/pre-mRNA binding were determined using a construct of CPSF30 that contains just the five CCCH domains (CPSF30-5F). Bioinformatics was utilized to identify the variability within the AU hexamer sequence in pre-mRNAs. The effects of this sequence variability on CPSF30-5F/RNA binding affinities were measured. Bases at positions 1, 2, 4, and 5 within the AU hexamer were found to be important for RNA binding. Bioinformatics revealed that the three bases flanking the AU hexamer at the 5' and 3' ends are twice as likely to be adenine or uracil as guanine and cytosine. The presence of A and U residues in these flanking regions was determined to promote higher-affinity CPSF30-5F/RNA binding than G and C residues. The addition of the zinc knuckle domain to CPSF30-5F (CPSF30-FL) restored binding to AU hexamer variants. This restoration of binding is connected to the presence of a U-rich sequence within the pre-mRNA to which the zinc knuckle binds. A mechanism of differential RNA binding by CPSF30, modulated by accessibility of the two RNA binding sites, is proposed.
Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Poli U/metabolismo , Poliadenilação , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Animais , Sítios de Ligação , Bovinos , Fator de Especificidade de Clivagem e Poliadenilação/química , Fator de Especificidade de Clivagem e Poliadenilação/genética , Ligação Proteica , Precursores de RNA/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Dedos de ZincoRESUMO
Coronaviruses (CoVs) are positive-sense RNA viruses that can emerge from endemic reservoirs and infect zoonotically, causing significant morbidity and mortality. CoVs encode an endoribonuclease designated EndoU that facilitates evasion of host pattern recognition receptor MDA5, but the target of EndoU activity was not known. Here, we report that EndoU cleaves the 5'-polyuridines from negative-sense viral RNA, termed PUN RNA, which is the product of polyA-templated RNA synthesis. Using a virus containing an EndoU catalytic-inactive mutation, we detected a higher abundance of PUN RNA in the cytoplasm compared to wild-type-infected cells. Furthermore, we found that transfecting PUN RNA into cells stimulates a robust, MDA5-dependent interferon response, and that removal of the polyuridine extension on the RNA dampens the response. Overall, the results of this study reveal the PUN RNA to be a CoV MDA5-dependent pathogen-associated molecular pattern (PAMP). We also establish a mechanism for EndoU activity to cleave and limit the accumulation of this PAMP. Since EndoU activity is highly conserved in all CoVs, inhibiting this activity may serve as an approach for therapeutic interventions against existing and emerging CoV infections.
Assuntos
Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Coronavirus/metabolismo , Endorribonucleases/metabolismo , Poli U/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Antivirais/farmacologia , Linhagem Celular , Chlorocebus aethiops , Coronavirus/enzimologia , Coronavirus/imunologia , Endorribonucleases/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Interferons/farmacologia , Poli U/química , RNA Viral/genética , RNA Viral/metabolismo , Uridina/química , Células Vero , Proteínas não Estruturais Virais/genética , Replicação Viral/fisiologiaRESUMO
Cleavage and polyadenylation specificity factor 30 (CPSF30) is a "zinc finger" protein that plays a crucial role in the transition of pre-mRNA to RNA. CPSF30 contains five conserved CCCH domains and a CCHC "zinc knuckle" domain. CPSF30 activity is critical for pre-mRNA processing. A truncated form of the protein, in which only the CCCH domains are present, has been shown to specifically bind AU-rich pre-mRNA targets; however, the RNA binding and recognition properties of full-length CPSF30 are not known. Herein, we report the isolation and biochemical characterization of full-length CPSF30. We report that CPSF30 contains one 2Fe-2S cluster in addition to five zinc ions, as measured by inductively coupled plasma mass spectrometry, ultraviolet-visible spectroscopy, and X-ray absorption spectroscopy. Utilizing fluorescence anisotropy RNA binding assays, we show that full-length CPSF30 has high binding affinity for two types of pre-mRNA targets, AAUAAA and polyU, both of which are conserved sequence motifs present in the majority of pre-mRNAs. Binding to the AAUAAA motif requires that the five CCCH domains of CPSF30 be present, whereas binding to polyU sequences requires the entire, full-length CPSF30. These findings implicate the CCHC "zinc knuckle" present in the full-length protein as being critical for mediating polyU binding. We also report that truncated forms of the protein, containing either just two CCCH domains (ZF2 and ZF3) or the CCHC "zinc knuckle" domain, do not exhibit any RNA binding, indicating that CPSF30/RNA binding requires several ZF (and/or Fe-S cluster) domains working in concert to mediate RNA recognition.
Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Poli U/metabolismo , Precursores de RNA/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Fator de Especificidade de Clivagem e Poliadenilação/química , Fator de Especificidade de Clivagem e Poliadenilação/genética , Cobalto/química , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Mutação , Ligação Proteica , Precursores de RNA/genética , Zinco/química , Dedos de Zinco , alfa-Sinucleína/genéticaRESUMO
Unlike double-stranded DNA, single-stranded RNA can be spontaneously packaged into spherical capsids by viral capsid protein (CP) because it is a more compact and flexible polymer. Many systematic investigations of this self-assembly process have been carried out using CP from cowpea chlorotic mottle virus, with a wide range of sequences and lengths of single-stranded RNA. Among these studies are measurements of the relative packaging efficiencies of these RNAs into spherical capsids. In this work, we address a fundamental issue that has received very little attention, namely the question of the preferred curvature of the capsid formed around different RNA molecules. We show in particular that homopolymers of RNA-polyribouridylic acid and polyriboadenylic acid-form exclusively T = 2-sized (â¼22-nm diameter) virus-like particles (VLPs) when mixed with cowpea chlorotic mottle virus CP, independent of their length, ranging from 500 to more than 4000 nucleotides. This is in contrast to "normal-composition" RNAs (i.e., molecules with comparable numbers of each of the four nucleotides and hence capable of developing a large amount of secondary structure because of intramolecular complementarity/basepairing); a curvature corresponding to T = 3-size (â¼28 nm in diameter) is preferred for the VLPs formed with such RNAs. Our work is consistent with the preferred curvature of VLPs being a consequence of interaction of CP with RNA-in particular, the presence or absence of short RNA duplexes-and suggests that the equilibrium size of the capsid results from a trade-off between this optimum size and the cost of confinement.
Assuntos
Bromovirus/química , RNA/química , Concentração de Íons de Hidrogênio , Poli A/química , Poli A/metabolismo , Poli U/química , Poli U/metabolismo , Polimerização , RNA/metabolismoRESUMO
Liquid-liquid phase separation (LLPS) of RNA-protein complexes plays a major role in the cellular function of membraneless organelles (MLOs). MLOs are sensitive to changes in cellular conditions, such as fluctuations in cytoplasmic ion concentrations. To investigate the effect of these changes on MLOs, we studied the influence of divalent cations on the physical and chemical properties of RNA coacervates. Using a model system comprised of an arginine-rich peptide and RNA, we predicted and observed that variations in signaling cations exert interaction-dependent effects on RNA LLPS. Changing the ionic environment has opposing effects on the propensity for heterotypic peptide-RNA and homotypic RNA LLPS, which results in a switch between coacervate types. Furthermore, divalent ion variations continuously tune the microenvironments and fluid properties of heterotypic and homotypic droplets. Our results may provide a general mechanism for modulating the biochemical environment of RNA coacervates in a cellular context.
Assuntos
Peptídeos/metabolismo , RNA/metabolismo , Sequência de Aminoácidos , Arginina/química , Cátions Bivalentes/química , Recuperação de Fluorescência Após Fotodegradação , Magnésio/química , Microscopia de Fluorescência , Nefelometria e Turbidimetria , Peptídeos/química , Poli U/química , Poli U/metabolismo , Ligação Proteica , RNA/químicaRESUMO
Phosphorylation mediated by protein kinases plays a pivotal role in metabolic and cell-signaling processes, and the dysfunction of protein kinases such as protein kinase A (PKA) may induce several human diseases. Therefore, it is of great significance to develop a facile and effective method for PKA activity assay and high-throughput screening of inhibitors. Herein, we develop a new fluorescent off-on method for PKA assay based on the assembly of anionic polyuridylic acid (polyU) and cationic fluorescent peptide. The phosphorylation of the peptide disrupts its electrostatic binding with polyU, suppresses the concentration quenching effect of polyU, and thus causes fluorescence recovery. The recovered fluorescence intensity at 585 nm is directly proportional to the PKA activity in the range of 0.1-3.2 U/mL with a detection limit of 0.05 U/mL. Using our method, the PKA activity in HeLa cell lysate is determined to be 58.2 ± 5.1 U/mg protein. The method has also been employed to evaluate the inhibitory effect of PKA inhibitors with satisfactory results and may be expected to be a promising candidate for facile and cost-effective assay of kinase activity and high-throughput inhibitor screening.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ensaios Enzimáticos/métodos , Corantes Fluorescentes/química , Peptídeos/química , Poli U/química , Trifosfato de Adenosina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Células HeLa , Humanos , Limite de Detecção , Peptídeos/metabolismo , Fosforilação , Poli U/metabolismo , Ligação Proteica , Espectrometria de FluorescênciaRESUMO
Translational control of gene expression plays essential roles in cellular stress responses and organismal development by enabling rapid, selective, and localized control of protein production. Translational regulation depends on context-dependent differences in the protein output of mRNAs, but the key mRNA features that distinguish efficiently translated mRNAs are largely unknown. Here, we comprehensively determined the RNA-binding preferences of the eukaryotic initiation factor 4G (eIF4G) to assess whether this core translation initiation factor has intrinsic sequence preferences that may contribute to preferential translation of specific mRNAs. We identified a simple RNA sequence motif-oligo-uridine-that mediates high-affinity binding to eIF4G in vitro. Oligo(U) motifs occur naturally in the transcript leader (TL) of hundreds of yeast genes, and mRNAs with unstructured oligo(U) motifs were enriched in immunoprecipitations against eIF4G. Ribosome profiling following depletion of eIF4G in vivo showed preferentially reduced translation of mRNAs with long TLs, including those that contain oligo(U). Finally, TL oligo(U) elements are enriched in genes with regulatory roles and are conserved between yeast species, consistent with an important cellular function. Taken together, our results demonstrate RNA sequence preferences for a general initiation factor, which cells potentially exploit for translational control of specific mRNAs.
Assuntos
Sítios de Ligação , Fator de Iniciação Eucariótico 4G/metabolismo , Regulação Fúngica da Expressão Gênica , Motivos de Nucleotídeos , Poli U/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequência Conservada , Ligação Proteica , Biossíntese de Proteínas , RNA Mensageiro/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Lin28a inhibits the biogenesis of let-7 miRNAs by triggering the polyuridylation and degradation of their precursors by terminal uridylyltransferases TUT4/7 and 3'-5' exoribonuclease Dis3l2, respectively. Previously, we showed that Lin28a also controls the production of neuro-specific miRNA-9 via a polyuridylation-independent mechanism. Here we reveal that the sequences and structural characteristics of pre-let-7 and pre-miRNA-9 are eliciting two distinct modes of binding to Lin28a. We present evidence that Dis3l2 controls miRNA-9 production. Finally, we show that the constitutive expression of untagged Lin28a during neuronal differentiation in vitro positively and negatively affects numerous other miRNAs. Our findings shed light on the role of Lin28a in differentiating cells and on the ways in which one RNA-binding protein can perform multiple roles in the regulation of RNA processing.
Assuntos
Exorribonucleases/genética , MicroRNAs/genética , Neurônios/metabolismo , Precursores de RNA/genética , Proteínas de Ligação a RNA/genética , Animais , Pareamento de Bases , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Exorribonucleases/metabolismo , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Camundongos , MicroRNAs/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Conformação de Ácido Nucleico , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Poli U/metabolismo , Ligação Proteica , Clivagem do RNA , RNA Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/metabolismo , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tretinoína/farmacologiaRESUMO
Cell-type-specific and inducible alternative splicing has a fundamental impact on regulating gene expression and cellular function in a variety of settings, including activation and differentiation. We have recently shown that activation-induced skipping of TRAF3 exon 8 activates noncanonical NF-κB signaling upon T cell stimulation, but the regulatory basis for this splicing event remains unknown. Here we identify cis- and trans-regulatory elements rendering this splicing switch activation dependent and cell type specific. The cis-acting element is located 340 to 440 nucleotides upstream of the regulated exon and acts in a distance-dependent manner, since altering the location reduces its activity. A small interfering RNA screen, followed by cross-link immunoprecipitation and mutational analyses, identified CELF2 and hnRNP C as trans-acting factors that directly bind the regulatory sequence and together mediate increased exon skipping in activated T cells. CELF2 expression levels correlate with TRAF3 exon skipping in several model systems, suggesting that CELF2 is the decisive factor, with hnRNP C being necessary but not sufficient. These data suggest an interplay between CELF2 and hnRNP C as the mechanistic basis for activation-dependent alternative splicing of TRAF3 exon 8 and additional exons and uncover an intronic splicing silencer whose full activity depends on the precise location more than 300 nucleotides upstream of the regulated exon.
Assuntos
Processamento Alternativo/genética , Proteínas CELF/metabolismo , Éxons/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/metabolismo , Íntrons/genética , Ativação Linfocitária/genética , Proteínas do Tecido Nervoso/metabolismo , Fator 3 Associado a Receptor de TNF/genética , Sítios de Ligação , Células HEK293 , Humanos , Poli U/metabolismo , Ligação Proteica/genética , RNA Interferente Pequeno/metabolismo , Elementos Silenciadores Transcricionais/genética , Linfócitos T/imunologia , Fator 3 Associado a Receptor de TNF/metabolismoRESUMO
RNA 3' uridylation occurs pervasively in eukaryotes, but is poorly characterized in viruses. In this study, we demonstrate that a broad array of RNA viruses, including mycoviruses, plant viruses and animal viruses, possess a novel population of RNA species bearing nontemplated oligo(U) or (U)-rich tails, suggesting widespread 3' uridylation in eukaryotic viruses. Given the biological relevance of 3' uridylation to eukaryotic RNA degradation, we propose a conserved but as-yet-unknown mechanism in virus-host interaction.
Assuntos
Poli U/metabolismo , Vírus de RNA/fisiologia , RNA Viral/metabolismo , Regiões 3' não Traduzidas , Animais , Eucariotos/virologia , Fungos , PlantasRESUMO
Uridylation emerges as a key modification promoting mRNA degradation in eukaryotes. In addition, uridylation by URT1 prevents the accumulation of excessively deadenylated mRNAs in Arabidopsis. Here, we show that the extent of mRNA deadenylation is controlled by URT1. By using TAIL-seq analysis, we demonstrate the prevalence of mRNA uridylation and the existence, at lower frequencies, of mRNA cytidylation and guanylation in Arabidopsis. Both URT1-dependent and URT1-independent types of uridylation co-exist but only URT1-mediated uridylation prevents the accumulation of excessively deadenylated mRNAs. Importantly, uridylation repairs deadenylated extremities to restore the size distribution observed for non-uridylated oligo(A) tails. In vivo and in vitro data indicate that Poly(A) Binding Protein (PABP) binds to uridylated oligo(A) tails and determines the length of U-extensions added by URT1. Taken together, our results uncover a role for uridylation and PABP in repairing mRNA deadenylated ends and reveal that uridylation plays diverse roles in eukaryotic mRNA metabolism.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , RNA Nucleotidiltransferases/metabolismo , RNA Mensageiro/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/genética , Sítios de Ligação , Western Blotting , Imunoprecipitação , MicroRNAs/metabolismo , Poli A/metabolismo , Poli U/metabolismo , Proteínas de Ligação a Poli(A)/genética , Interferência de RNA , RNA Nucleotidiltransferases/genética , Estabilidade de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
UNLABELLED: Despite the introduction of direct-acting antiviral (DAA) drugs against hepatitis C virus (HCV), infection remains a major public health concern because DAA therapeutics do not prevent reinfection and patients can still progress to chronic liver disease. Chronic HCV infection is supported by a variety of viral immune evasion strategies, but, remarkably, 20% to 30% of acute infections spontaneously clear prior to development of adaptive immune responses, thus implicating innate immunity in resolving acute HCV infection. However, the virus-host interactions regulating acute infection are unknown. Transmission of HCV involves one or a few transmitted/founder (T/F) variants. In infected hepatocytes, the retinoic acid-inducible gene I (RIG-I) protein recognizes 5' triphosphate (5'ppp) of the HCV RNA and a pathogen-associated molecular pattern (PAMP) motif located within the 3' untranslated region consisting of poly-U/UC. PAMP binding activates RIG-I to induce innate immune signaling and type 1 interferon antiviral defenses. HCV poly-U/UC sequences can differ in length and complexity, suggesting that PAMP diversity in T/F genomes could regulate innate immune control of acute HCV infection. Using 14 unique poly-U/UC sequences from HCV T/F genomes recovered from acute-infection patients, we tested whether RIG-I recognition and innate immune activation correlate with PAMP sequence characteristics. We show that T/F variants are recognized by RIG-I in a manner dependent on length of the U-core motif of the poly-U/UC PAMP and are recognized by RIG-I to induce innate immune responses that restrict acute infection. PAMP recognition of T/F HCV variants by RIG-I may therefore impart innate immune signaling and HCV restriction to impact acute-phase-to-chronic-phase transition. IMPORTANCE: Recognition of nonself molecular patterns such as those seen with viral nucleic acids is an essential step in triggering the immune response to virus infection. Innate immunity is induced by hepatitis C virus infection through the recognition of viral RNA by the cellular RIG-I protein, where RIG-I recognizes a poly-uridine/cytosine motif in the viral genome. Variation within this motif may provide an immune evasion strategy for transmitted/founder viruses during acute infection. Using 14 unique poly-U/UC sequences from HCV T/F genomes recovered from acutely infected HCV patients, we demonstrate that RIG-I binding and activation of innate immunity depend primarily on the length of the uridine core within this motif. T/F variants found in acute infection contained longer U cores within the motif and could activate RIG-I and induce innate immune signaling sufficient to restrict viral infection. Thus, recognition of T/F variants by RIG-I could significantly impact the transition from acute to chronic infection.
Assuntos
RNA Helicases DEAD-box/metabolismo , Variação Genética , Hepacivirus/genética , Hepatite C/imunologia , Imunidade Inata/imunologia , Poli U/metabolismo , Linhagem Celular , Proteína DEAD-box 58 , Ensaio de Desvio de Mobilidade Eletroforética , Hepacivirus/metabolismo , Hepatócitos/metabolismo , Hepatócitos/virologia , Interações Hospedeiro-Patógeno , Humanos , Immunoblotting , Plasmídeos/genética , Poli U/genética , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , Receptores ImunológicosRESUMO
Simple nucleotide templating activities are of interest as potential primordial reactions. Here we describe the acceleration of 5'-5' AppA synthesis by 3'-5' poly(U) under normal solution conditions. This reaction is apparently templated via complementary U:A base-pairing, despite the involvement of two different RNA backbones, because poly(U), unlike other polymers, significantly stimulates AppA synthesis. These interactions occur in moderate (K(+)) and (Mg(2+)) and are temperature sensitive, being more efficient at 10°C than at 4°C, but absent at 20°C. The reaction is only slightly pH sensitive, despite potentially relevant substrate pKa's. Kinetic data explicitly support production of AppA by interaction of stacked 2MeImpA and pA nucleotides paired with a single molecule of U template. At a lower rate, AppA can also be produced by a chemical reaction between 2MeImpA and pA, without participation of poly(U). Molecular modeling suggests that 5'-5' joining between stacked or concurrently paired A's can occur without major departures from normal U-A helical coordinates. So, coenzyme-like 5'-5' purine dinucleotides might be readily synthesized from 3'-5' RNAs with complementary sequences.
Assuntos
Fosfatos de Dinucleosídeos/biossíntese , Poli U/metabolismo , RNA/metabolismoRESUMO
RNA molecules are subjected to post-transcriptional modifications that might determine their maturation, activity, localization and stability. These alterations can occur within the RNA molecule or at its 5'- or 3'- extremities, and are essential for gene regulation and proper function of the RNA. One major type of modification is the 3'-end addition of nontemplated nucleotides. Polyadenylation is the most well studied type of 3'-RNA modification, both in eukaryotes and prokaryotes. The importance of 3'-oligouridylation has recently gained attention through the discovery of several types of uridylated-RNAs, by the existence of enzymes that specifically add poly(U) tails and others that preferentially degrade these tails. Namely, Dis3L2 is a 3'-5' exoribonuclease from the RNase II/RNB family that has been shown to act preferentially on oligo(U)-tailed transcripts. Our understanding of this process is still at the beginning, but it is already known to interfere in the regulation of diverse RNA species in most eukaryotes. Now that we are aware of the prevalence of RNA uridylation and the techniques available to globally evaluate the 3'-terminome, we can expect to make rapid progress in determining the extent of terminal oligouridylation in different RNA populations and unravel its impact on RNA decay mechanisms. Here, we sum up what is known about 3'-RNA modification in the different cellular compartments of eukaryotic cells, the conserved enzymes that perform this 3'-end modification and the effectors that are selectively activated by this process.
Assuntos
Processamento de Terminações 3' de RNA , RNA/química , RNA/metabolismo , Animais , Compartimento Celular , Exorribonucleases/química , Exorribonucleases/metabolismo , Humanos , Redes e Vias Metabólicas , Modelos Biológicos , Modelos Moleculares , Oligorribonucleotídeos/química , Oligorribonucleotídeos/metabolismo , Poli U/química , Poli U/metabolismo , Conformação Proteica , Estabilidade de RNA , Nucleotídeos de Uracila/química , Nucleotídeos de Uracila/metabolismoRESUMO
Erb1 (Eukaryotic Ribosome Biogenesis 1) protein is essential for the maturation of the ribosomal 60S subunit. Functional studies in yeast and mammalian cells showed that altogether with Nop7 and Ytm1 it forms a stable subcomplex called PeBoW that is crucial for a correct rRNA processing. The exact function of the protein within the process remains unknown. The N-terminal region of the protein includes a well conserved region shown to be involved in PeBoW complex formation whereas the carboxy-terminal half was predicted to contain seven WD40 repeats. This first structural report on Erb1 from yeast describes the architecture of a seven-bladed ß-propeller domain that revealed a characteristic extra motif formed by two α-helices and a ß-strand that insert within the second WD repeat. We performed analysis of molecular surface and crystal packing, together with multiple sequence alignment and comparison of the structure with other ß-propellers, in order to identify areas that are more likely to mediate protein-protein interactions. The abundance of many positively charged residues on the surface of the domain led us to investigate whether the propeller of Erb1 might be involved in RNA binding. Three independent assays confirmed that the protein interacted in vitro with polyuridilic acid (polyU), thus suggesting a possible role of the domain in rRNA rearrangement during ribosome biogenesis.
Assuntos
RNA/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Cristalografia por Raios X , Evolução Molecular , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Poli U/química , Poli U/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , RNA/química , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
There is renewed interest in investigating triplex nucleic acids because triplexes may be implicated in a range of cellular functions. However, the stabilization of triplex nucleic acids is essential to achieve their biological functions. In contrast to triplex DNA, little has been reported concerning the recognition of triplex RNA by transition-metal complexes at present. We report here a ruthenium(ii) polypyridyl complex, [Ru(bpy)2(mdpz)](2+) (bpy = 2,2'-bipyridine; mdpz = 7,7'-methylenedioxyphenyl-dipyrido-[3,2-a:2',3'-c]phenazine), as a sensitive luminescent probe for poly(U)·poly(A)*poly(U), which can strongly stabilize the triplex RNA from 37.5 to 53.1 °C in solution. The main results further advance our knowledge on the triplex RNA-binding by metal complexes, particularly ruthenium(ii) complexes.
Assuntos
2,2'-Dipiridil/análogos & derivados , Compostos Organometálicos/farmacologia , Estabilidade de RNA/efeitos dos fármacos , RNA/química , RNA/metabolismo , 2,2'-Dipiridil/farmacologia , Conformação de Ácido Nucleico , Poli A/química , Poli A/metabolismo , Poli U/química , Poli U/metabolismo , Temperatura de Transição , ViscosidadeRESUMO
Studies on the molecular aspects of alkaloid-RNA complexation are of prime importance for the development of rational RNA targeted drug design strategies. Towards this goal, the binding aspects of three novel 9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberine analogs to four single stranded ribonucleotides, poly(G), poly(I), poly(C) and poly(U), were studied for the first time employing multifaceted biophysical tools. Absorbance and fluorescence studies revealed that these analogs bound non-cooperatively to poly(G) and poly(I) with binding affinities remarkably higher than berberine. The binding of these analogs to poly(U) and poly(C) was weaker in comparison to poly(G) and poly(I) but were one order higher in comparison to berberine. Quantum efficiency values revealed that energy transfer occurred from the RNA bases to the analogs upon complexation. The binding was dominated by large positive entropic contributions and small but favorable enthalpic contributions. Salt dependent studies established that the binding was dominated by hydrophobic forces that contributed around 90% of the total standard molar Gibbs energy. The chain length of the substitution at the 9-position was found to be critical in modulating the binding affinities. These results provide new insights into the binding efficacy of these novel berberine analogs to single stranded RNA sequences.
Assuntos
Berberina/análogos & derivados , Ribonucleotídeos/química , Berberina/metabolismo , Sítios de Ligação , Calorimetria , Dicroísmo Circular , Conformação de Ácido Nucleico , Concentração Osmolar , Poli C/química , Poli C/metabolismo , Poli G/química , Poli G/metabolismo , Poli I/química , Poli I/metabolismo , Poli U/química , Poli U/metabolismo , Teoria Quântica , RNA/química , RNA/metabolismo , Ribonucleotídeos/metabolismo , Espectrometria de Fluorescência , Temperatura , TermodinâmicaRESUMO
Eukaryotic translation initiation factor eIF4A is a DEAD-box helicase that resolves secondary structure elements in the 5'-UTR of mRNAs during ribosome scanning. Its RNA-stimulated ATPase and ATP-dependent helicase activities are enhanced by other translation initiation factors, but the underlying mechanisms are unclear. DEAD-box proteins alternate between open and closed conformations during RNA unwinding. The transition to the closed conformation is linked to duplex destabilization. eIF4A is a special DEAD-box protein that can adopt three different conformations, an open state in the absence of ligands, a half-open state stabilized by the translation initiation factor eIF4G and a closed state in the presence of eIF4G and eIF4B. We show here that eIF4A alone does not measurably sample the closed conformation. The translation initiation factors eIF4B and eIF4G accelerate the eIF4A conformational cycle. eIF4G increases the rate of closing more than the opening rate, and eIF4B selectively increases the closing rate. Strikingly, the rate constants and the effect of eIF4B are different for different RNAs, and are related to the presence of single-stranded regions. Modulating the kinetics of the eIF4A conformational cycle is thus central for the multi-layered regulation of its activity, and for its role as a regulatory hub in translation initiation.